LOCUS       sequence-224565-        5641 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses IMX-DogTag in bacterial cells for modular antigen 
            multimerization using SnoopLigase.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5641)
  AUTHORS   Andersson AC, Buldun CM, Pattinson DJ, Draper SJ, Howarth M
  TITLE     SnoopLigase peptide-peptide conjugation enables modular vaccine 
            assembly.
  JOURNAL   Sci Rep. 2019 Mar 15;9(1):4625. doi: 10.1038/s41598-019-40985-w.
  PUBMED    30874593
REFERENCE   2  (bases 1 to 5641)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Sci Rep."; 
            date: "2019-03-15"; volume: "9(1):4625. doi"; pages: " 
            10.1038/s41598-019-40985-w"
COMMENT     Sequence Label: pET28a-IMX-DogTag
FEATURES             Location/Qualifiers
     source          1..5641
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     2..21
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     105..124
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        105..123
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    124..148
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     RBS             163..185
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             396..464
                     /codon_start=1
                     /product="peptide derived from the RrgA adhesin of 
                     Streptococcus pneumoniae"
                     /label=DogTag
                     /note="forms a covalent bond with the DogCatcher protein 
                     when inserted into an intra-domain protein loop (Keeble et 
                     al., 2022)"
                     /translation="DIPATYEFTDGKHYITNEPIPPK"
     CDS             477..494
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(547..565)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      561..608
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      645..1100
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(732..751)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     942..963
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1126..1230
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1231..1299,1300..2091)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1449..1468)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2262..2850
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2751..2770
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3004..3021
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3036..3175
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3101..3120)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3261..3283
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3277..3468)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4277..5359)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     5321..5340
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5360..5437)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 cggtgatgtc ggcgatatag gcgccagcaa ccgcacctgt ggcgccggtg atgccggcca
       61 cgatgcgtcc ggcgtagagg atcgagatct cgatcccgcg aaattaatac gactcactat
      121 aggggaattg tgagcggata acaattcccc tctagaaata attttgttta actttaagaa
      181 ggagatatac catgggcagc agcaaaaaac aaggtgatgc agatgtttgt ggcgaagtcg
      241 cctatatcca atccgttgtt agtgattgtc acgtaccaac tgccgaattg cgtacccttt
      301 tggagatccg taagctgttt cttgagattc aaaaactgaa agttgaatta cagggtctta
      361 gcaaggaagg ttccggatcc ggtgaaggtt ccggtgatat tccggctaca tatgaattta
      421 ccgatggtaa acattatatc accaatgaac cgataccgcc gaaataataa ctcgagcacc
      481 accaccacca ccactgagat ccggctgcta acaaagcccg aaaggaagct gagttggctg
      541 ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg gtcttgaggg
      601 gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc cctgtagcgg
      661 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
      721 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
      781 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
      841 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
      901 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
      961 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     1021 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     1081 aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     1141 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctggtaa
     1201 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     1261 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     1321 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     1381 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     1441 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     1501 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     1561 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     1621 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     1681 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     1741 ataccaaacg acgagcgtga caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa
     1801 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     1861 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     1921 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
     1981 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     2041 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     2101 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     2161 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     2221 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     2281 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     2341 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     2401 aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     2461 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     2521 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     2581 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     2641 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     2701 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     2761 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     2821 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     2881 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     2941 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
     3001 cgcagcgagt cagtgagcga ggaagcggaa gagcgcctga tgcggtattt tctccttacg
     3061 catctgtgcg gtatttcaca ccgcaatggt gcactctcag tacaatctgc tctgatgccg
     3121 catagttaag ccagtataca ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg
     3181 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
     3241 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
     3301 gaaacgcgcg aggcagctgc ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat
     3361 gtctgcctgt tcatccgcgt ccagctcgtt gagtttctcc agaagcgtta atgtctggct
     3421 tctgataaag cgggccatgt taagggcggt tttttcctgt ttggtcactg atgcctccgt
     3481 gtaaggggga tttctgttca tgggggtaat gataccgatg aaacgagaga ggatgctcac
     3541 gatacgggtt actgatgatg aacatgcccg gttactggaa cgttgtgagg gtaaacaact
     3601 ggcggtatgg atgcggcggg accagagaaa aatcacacag ggtcaatgcc agcgcttcgt
     3661 taatacagat gtaggtgttc cacagggtag ccagcagcat cctgcgatgc agatccggaa
     3721 cataatggtg cagggcgctg acttccgcgt ttccagactt tacgaaacac ggaaaccgaa
     3781 gaccattcat gttgttgctc aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg
     3841 ctcgcgtatc ggtgattcat tctgctaacc agtaaggcaa ccccgccagc ctagccgggt
     3901 cctcaacgac aggagcacga tcatgcgcac ccgtggggcc gccatgccgg cgataatggc
     3961 ctgcttctcg ccgaaacgtt tggtggcggg accagtgacg aaggcttgag cgagggcgtg
     4021 caagattccg aataccgcaa gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc
     4081 ctcgccgaaa atgacccaga gcgctgccgg cacctgtcct acgagttgca tgataaagaa
     4141 gacagtcata agtgcggcga cgatagtcat gccccgcgcc caccggaagg agctgactgg
     4201 gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc taatgagtga gctaacttac
     4261 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     4321 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt
     4381 ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga
     4441 gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg
     4501 ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatat
     4561 ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat
     4621 cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt
     4681 gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc
     4741 gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc
     4801 ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg
     4861 taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa
     4921 ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg
     4981 gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac
     5041 aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg
     5101 cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg
     5161 caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt
     5221 aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg
     5281 cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt
     5341 ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg
     5401 ccataccgcg aaaggttttg cgccattcga tggtgtccgg gatctcgacg ctctccctta
     5461 tgcgactcct gcattaggaa gcagcccagt agtaggttga ggccgttgag caccgccgcc
     5521 gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc ccccggccac ggggcctgcc
     5581 accataccca cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg atcttcccca
     5641 t
//
